Information on the amino acid sequence of naturally obtained peptides and proteins is essential in studies on the biological properties and functions thereof. Currently, all amino acid sequences of peptides and proteins are determined from the corresponding genetic information, namely, estimated on the basis of sequence of the genomic genes or c-DNAs prepared from m-RNAs coding these peptides. The amino acid sequence of proteins is also determined directly by various methods, alternatively. Information on partial amino acid sequence of a peptide is nevertheless needed in specifying a genomic gene or a c-DNA prepared from m-RNA coding the peptide.
Information on N- and C-terminal amino acid sequences of peptide is considered particularly useful as the information on the partial amino acid sequence of the peptide. For example if N-terminal amino acid sequence and C-terminal amino acid sequence are available, in selecting a c-DNA coding an analyte peptide from a c-DNA library prepared from a number of m-RNAs, it becomes possible to prepare a nucleic acid probe based on the amino acid sequences at both terminals and select a desirable c-DNA by using the probe obtained. It is also possible to amplify a desirable c-DNA selectively by applying the Polymerase Chain Reaction (PCR) method, by using the oligonucleotide primer prepared based on the amino acid sequences at both terminals.
A method of degrading N-terminal amino acids sequentially by using Edman degradation and identifying the amino acid derivatives produced has been conventionally used in analyzing the N-terminal amino acid sequence of a peptide.
On the other hand, methods of sequentially degrading C-terminal amino acids chemically and specifying the degraded C-terminal amino acids from the difference in molecular weight between shortened peptides obtained as the reaction products, and the original peptide has been proposed as a means which analyzes the C-terminal amino acid sequence of the peptide (see, Non-Patent Documents 1, 2, and 3).
Non-Patent Document 1 discloses a method of degrading C-terminal amino acids sequentially by a chemical method. It is a method of accelerating selective hydrolysis of C-terminal amino acids by heating a dry peptide to 90° C. and allowing a vapor from a high-concentration aqueous solution of pentafluoropropanoic acid (CF3CF2COOH) or heptafluorobutanoic acid (CF3CF2CF2COOH) to act on the peptide.
Alternatively, Non-Patent Documents 2 and 3 disclose methods of degrading C-terminal amino acids selectively by using an acetonitrile solution of pentafluoropropanoic anhydride ((CF3CF2CO)2O) or heptafluorobutanoic anhydride ((CF3CF2CF2CO)2O) instead of the high-concentration aqueous solution of perfluoroalkanoic acid. It is reported that generation of the side reactions could be avoided, for example, by allowing the vapor of the solution to act on a dry peptide while cooling the solution to −18° C. and thus, preventing penetration of the water molecules vaporized from the solution into the system.
In these conventional C-terminal degradation methods, an oxazolone ring structure is seemingly formed as a reaction intermediate from the C-terminal amino acid in the dehydration reaction represented by the reaction formula (I) below. The reaction represented by the reaction formula (II) below occurs then in reaction of perfluoroalkanoic acid with the oxazolone ring. As a result of this, it is reported that the C-terminal amino acid is achieved to be degraded selectively.
The selective degradation reaction of C-terminal amino acids proceeds sequentially, giving a mixture containing a series of reaction products in which one to ten or more amino acid residues have been deleted from the C-terminal of the original peptide when a predetermined processing period. When the mass of the ionic species derived from respective reaction products is measured by analyzing the mixture containing a series of reaction products by mass spectrometry, a series of peaks showing the difference in mass reflecting the C-terminal amino acid sequence are measured.
For example, each of reaction products generated by sequential degradation reaction of C-terminal amino acid from the original peptide is a group of a series of reaction products of several types, up to reaction products in which several amino acid residues have been deleted from the original peptide. It is possible to analyze the mass of the corresponding ionic species all together by subjecting the reaction products to analysis by mass spectrometry. It is considered possible to determine the C-terminal amino acid sequence over several amino acid residues collectively from the mass of the ionic species corresponding to the deleted C-terminal-sided amino acids.
    Non-Patent Document 1: Tsugita, A. et al., Eur. J. Biochem., 1992, 206, p. 691-696    Non-Patent Document 2: Tsugita, A. et al., Chem. Lett., 1992, p. 235-238    Non-Patent Document 3: Takamoto, K. et al., Eur. J. Biochem. 228, 1995, p. 362-372